The present study set up a multi-stage gut simulator (MS-GUTS) system, which was used to study gut microbiota. Analyses of the bacterial communities in Vessels 4, 5, and 6 that represent ascending colon, transverse colon, and descending colon, respectively, using denaturing gradient gel electrophoresis (DGGE) showed that a steady state bacterial community could be established after inoculation of the bioreactor vessels with human faecal material. To develop analytical methods, a mock bacterial community was analysed by DGGE, qPCR and 16S rRNA community profiling methods. Results showed that DGGE is not applicable in quantitative analysis due to the method’s limitation, while qPCR and the 16S rRNA microbiome profiling analysis provided results consistent with that of the expected values. After optimizing the MS-GUTS platform and the analytical tools, two application studies were performed. First, the effect of resveratrol on gut microbiota was investigated. Results showed no statistically significant change to the bacterial groups due to the large variability in gut microbiota’s relative abundance. Two ecological parameters, namely indices of diversity and evenness, were found to be significantly different between before and after experimental treatment in Vessel 4. In the second application study of microbial interactions, the MS-GUTS was used to investigate quorum sensing and gene transfer. Results showed no detection of quorum sensing molecule (autoinducer-2) in the mixed microbial community. In the gene transfer study, an exogenous E. coli O157:H7 carrying a plasmid containing a lux gene was introduced into the system, functioning as a gene donor. Bioluminescence was used to monitor gene transfer. An unknown luminescent bacterium was isolated, and identified as Pseudomonas aeruginosa. Finally, as the MS-GUTS is limited by its absence of host tissue component, a proposed modification to include organ baths was attempted. Porcine intestinal tissue segments were integrated into Vessels 3 and 6, representing ileum and descending colon, respectively. Results showed temporary viability of the intestinal tissues of approximately 4-5 h, which challenged the application of the set up. In conclusion, the present study successfully set up the MS-GUTS system as well as performed two application studies to investigate the gut microbiota.